The neurotrophins, including nerve growth factor (NGF), brain derived neutrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5), are among the molecular determinants that regulate the generation of diverse neuronal populations and the maintenance of their functions within the nervous system (Lewin G R, et al. 1996. Ann. Rev. Neurosci. 19:289; Theonen H. 1995. Science 70:593). The binding of mature neurotrophins to their cognate receptor tyrosine kinase, trks, (i.e., NGF to trk A, BDNF and NT-4/5 to trk B, and NT-3 to trk C) triggers a cascade of anti-apoptotic events that ensure neuronal survival. In competition analysis, the mature form of NGF is more potent than the larger pro-forms of NGF in trk A binding (Chen Y E, et al. 1997. Mol. Cell Endocrinol. 127:129) and in promoting cell survival.
In addition to trks, all mature neurotrophins are thought to interact with the p75 receptor. The p75 receptor belongs to the tumor necrosis factor (TNF) receptor superfamily. The extracellular domain of p75 that is the most critical for neurotrophin binding is reported to be the third and fourth cysteine loops (Choi D W. 1990. J. Neurosci. 10:2493; Yan H, et al. 1991. J. Biol. Chem. 266:12099).
The mechanism of signaling elicited by activated trks is unequivocal. However, the molecular and biological consequences of mature neurotrophin-p75 interaction have been confounding.
For example, structural analysis indicates that multiple regions of the extracellular domain of the p75 receptor contribute to mature NGF binding. Point mutagenesis suggests that the variable loops of mature neurotrophins are most important for interaction with p75 (Ryden M, et al. 1995. Embo J. 14:1979). Despite their importance for binding, these variable loops exhibit the highest degree of variation between the different mature neurotrophins. Nevertheless, all mature neurotrophins bind to p75.
Similarly, kinetic analysis of mature NGF-p75 binding demonstrates that mature NGF binds very rapidly, and dissociates very rapidly (Mahadeo D L, et al. 1994. J. Biol. Chem. 269:6884; Tait J F, et al. 1981. J. Biol. Chem. 256:11086). These results are perplexing, since most ligand-receptor interactions exhibit slow dissociation rates. In contrast, mature NGF binds to trk A slowly and is released very slowly.
When both types of receptors are present in the same cell, p75 is reported to enhance the affinity of trk A towards mature NGF (Mahadeo D L, et al., 1994. J. Biol. Chem. 269:6884; Hempstead, B. L., et al. 1991. Nature 350:678-683). In the absence of trk, however, binding of mature neurotrophin to p75 is reported to induce apoptosis (Chao M V, et al., 2001. Curr. Opin. Neurobiol. 11:281-286.). In addition, apoptosis is believed to be induced in neuronal cells via p75 when mismatching of mature neurotrophin-trk pairing occurs (Majdan M. 1999. Int J. Dev. Neurosci. 17:153).
For example, superior cervical ganglionic neurons express trk A and p75, but not trk B. As mentioned above, it is known that mature BDNF binds to trk B and to p75, but not to trk A. As a result of the mismatching of mature neurotrophin-trk pairing, treatment of superior cervical ganglionic neurons with mature BDNF triggers p75-mediated apoptosis (Bamji S X, et al. 1998. J. Cell. Biol. 140:911). Therefore, apoptosis is believed to be induced in neuronal cells when p75 is expressed alone. In addition, when both p75 and trk are present in the same cells, apoptosis is induced during mismatching of mature neurotrophin-trk pairing or when trk mediated signaling is inhibited.
Accordingly, currently available data have refined the original neurotrophin hypothesis. The original hypothesis stated that the lack of trophic support leads to selective neuronal loss (Korsching S. 1993. J. Neurosci. 13:2739). The refined model states that inappropriate activation of p75 by mature neurotrophins will lead to apoptosis.
If the refined model proves to be correct, the clinical implications are very significant. The p75 receptor is up-regulated in neuronal populations after nervous system stress or injury (Roux P P, et al., 1999. J. Neurosci. 19:6887; Widenfalk J, et al. 2001. J. Neurosci. 21:3457; van Eden C G, et al. 1994. Brain Res. Dev. Brain Res. 82:167) and in glia cells of multiple sclerosis patients (Dowling P, et al. 1999. Neurology 53:1676; Chang A, et al. 2000. J. Neurosci. 20:6404). Therefore, under the refined model, activation of p75 by mature neurotrophins in these cells will lead to apoptosis.
Recent findings have implicated apoptosis as a common endpoint of various nervous system injuries and environmental insults. Dying neurons in selected instances of, for example, hypoxic ischemia (Choi D W. 1990. J. Neurosci. 10:2493), viral infection (Lin K I, et al. 1996. J. Cell Biol. 131:1149), and neurodegenerative disorders (Roy N, et al. 1995. Cell 80:167; Liston P, et al. 1996. Nature 379:349) display a number of hallmark characteristics of apoptotic cells.
There is increasing evidence that mature neurotrophins and their receptors (p75 and trks) can also modulate a variety of cellular events in non-neuronal tissues (Lomen-Hoerth C, et al. 1995. J. Neurochem. 64:1780). In some instances, p75 expression is distinct from that of the trks (Wheeler E F, et al. 1998. J. Comp Neurol. 4:407). The distinct expression supports a trk-independent mechanism for p75 function in non-neuronal cells.
For example, NGF and p75 immunoreactivites have been detected in developing incisors (Mitsiadis T A, et al. 1993. Differentiation 54:161). In human adults, upregulation of p75 in renal biopsies is correlated with various glomerulopathies. The increased expression of p75 detected in renal biopsies apparently recapitulates a normal developing state during kidney development. Both p75 and NGF have been detected in developing muscles. In vitro analysis of the muscle cell line C2C12 suggests that p75-mediated myogenic differentiation is inhibited by NGF. The inhibition of myogenic differention by the NGF-p75 interaction occurs in a trk-independent manner. The expression of p75 is also detected in the atherosclerotic plaques of smooth muscle cells, as well as in many carcinoma cell types.
In addition, the expression of p75 has been observed in hair follicles (Yardley G, et al. 2000. Exp. Dermatol. 9:283; Botchkarev V A, et al. 2000. FASEB J. 14:1931). Both p75 and trks are found in hair follicles in the anagen (active growth) phase of hair growth (Botchkarev V A, et al. 2000. FESEB J. 14:1931). However, during the catagen (regression) phase, there is a disappearance of trk A, trk B, and trk C. The expression of p75 alone is markedly upregulated and detected in cells undergoing apoptosis (Botchkarev V A, et al. 2000. FESEB J. 14:1931). These findings suggest that p75 is involved in both the survival and apoptosis of cells in the hair follicle.
The interaction of p75 and neurotrophins in both neuronal and non-neuronal cells play a very important role in normal tissue development, as well as in many disease states. Currently available data suggest that p75 promotes both cell survival and apoptosis. However, conflicting and perplexing data in the prior art, such as the data described above, would have precluded the consideration of modulating mature neurotrophin-p75 interaction as a practical means for treating particular disorders.
Unwanted cell growth is a problem in various hyperproliferative conditions, such as cancer, acne, shingles, etc. Unwanted cell death is a problem in other conditions, such as injuries due to trauma, hair loss, etc. Thus, there is an immediate need for modulating apoptosis.
In certain instances, it is desirable to promote the p75-mediated apoptosis of deleterious cells. In other instances, it is desirable to inhibit the p75-mediated apoptosis (for example, in dying neurons and in dying hair follicles) without affecting the survival promoting functions of mature neurotrophins.